The T cell receptor (TCR) interacts with peptide-major histocompatibility complex (pMHC) to enable T cell development and trigger adaptive immune responses. For this reason, TCR:pMHC interactions have been intensely studied for over two decades. However, the details of how various binding parameters impact T cell activation remain elusive. Most measurements were made using recombinant proteins by surface plasmon resonance, a three-dimensional (3D) technique in which fluid-phase receptors and ligands are removed from their cellular environment. This approach found TCR:pMHC interactions with relatively low affinities and slow off-rates for agonist peptides. Newer generation techniques have analyzed TCR:pMHC interactions in two dimensions (2D), with both proteins anchored in apposing plasma membranes. These approaches reveal in situ TCR:pMHC interaction kinetics that are of high affinity and exhibit rapid on- and off-rates upon interaction with agonist ligands. Importantly, 2D binding parameters correlate better with T cell functional responses to a spectrum of ligands than 3D measures.

T cells recognizing self-peptides that mediate autoimmune disease and those that are responsible for efficacious immunity against pathogens may differ in affinity for antigen due to central and peripheral tolerance mechanisms. Here we utilize prototypical self-reactive (myelin) and viral-specific (LCMV) T cells from T cell receptor (TCR) transgenic mice (2D2 and SMARTA, respectively) to explore affinity differences. The T cells responsive to virus possessed >10,000 fold higher 2D affinity as compared to the self-reactive T cells. Despite their dramatically lower affinity for their cognate ligand, 2D2 T cells respond with complete, albeit delayed, activation (proliferation and cytokine production). SMARTA activation occurs rapidly, achieving peak phosphorylation of p38 (1 minute), Erk (30 minutes), and Jun (3 hours) as well as CD69 and CD25 upregulation (3 and 6 hours, respectively), with a corresponding early initiation of proliferation. 2D2 stimulation with MOG results in altered signaling – no phospho-Erk or phospho-p38 accumulation, significantly delayed activation kinetics of Jun (12 hours), and delayed but sustained SHP-1 activity – as well as delayed CD69 and CD25 expression (12–24 hours), and slow initiation of proliferation. This delay was not intrinsic to the 2D2 T cells, as a more potent antigen with >100-fold increased 2D affinity restored rapid response kinetics in line with those identified for the viral antigen. Taken together, these data demonstrate that time can offset low TCR affinity to attain full activation and suggest a mechanism by which low affinity T cells participate in autoimmune disease.

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus.

T cells rapidly undergo contraction upon viral clearance, but how T cell function and fate are determined during this phase is unclear. During the contraction phase of an acute infection with lymphocytic choriomeningitis virus, we found that virus-specific CD8 + T cells within the splenic red pulp (RP) had higher two-dimensional (2D) effective affinity than those within the white pulp (WP). This increased antigen recognition of RP-derived CD8 + T cells correlated with more efficient target cell killing and improved control of viremia. FoxP3 + regulatory T cells and cytokine TGF-β limited the 2D-affinity in the WP during the contraction phase. Anatomical location drove gene expression patterns in CD8 + T cells that led to preferential differentiation of memory precursor WP T cells into long-term memory cells. These results highlight that intricate regulation of T cell function and fate is determined by anatomic compartmentalization during the early immune contraction phase.

Seeding nerve guidance conduits with Schwann cells can improve the outcome of peripheral nerve injury repair. Bone marrow stem cells (BMSCs) represent a good choice of cell source as they can differentiate into Schwann cells under appropriate conditions. In this work, we systematically investigated the differentiation of BMSCs into Schwann cells on scaffolds comprising electrospun fibers. We changed the alignment, diameter, and surface properties of the fibers to optimize the differentiation efficiency. The uniaxial alignment of fibers not only promoted the differentiation of BMSCs into Schwann cells but also dictated the morphology and alignment of the derived cells. Coating the surface of aligned fibers with laminin further enhanced the differentiation and thus increased the secretion of neurotrophins. When co-cultured with PC12 cells or chick dorsal root ganglion, the as-derived Schwann cells were able to promote the outgrowth of neurites from cell bodies and direct their extension along the fibers, demonstrating the positive impacts of both the neurotrophic effect and the morphological contact guidance. This work offers a promising strategy for integrating fiber guidance with stem cell therapy to augment peripheral nerve injury repair.

Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes' tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP's force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.

Cells in the body are actively engaging with their environments that include both biochemical and biophysical aspects. The process by which cells convert mechanical stimuli from their environment to intracellular biochemical signals is known as mechanotransduction. Exemplifying the reliance on mechanotransduction for their development, differentiation and function are T cells, which are central to adaptive immune responses. T cell mechanoimmunology is an emerging field that studies how T cells sense, respond and adapt to the mechanical cues that they encounter throughout their life cycle. Here we review different stages of the T cell's life cycle where existing studies have shown important effects of mechanical force or matrix stiffness on a T cell as sensed through its surface molecules, including modulating receptor-ligand interactions, inducing protein conformational changes, triggering signal transduction, amplifying antigen discrimination and ensuring directed targeted cell killing. We suggest that including mechanical considerations in the immunological studies of T cells would inform a more holistic understanding of their development, differentiation and function.

TCRs recognize cognate pMHCs to initiate T cell signaling and adaptive immunity. Mechanical force strengthens TCR-pMHC interactions to elicit agonist-specific catch bonds to trigger TCR signaling, but the underlying dynamic structural mechanism is unclear. We combined steered molecular dynamics (SMD) simulation, single-molecule biophysical approaches, and functional assays to collectively demonstrate that mechanical force induces conformational changes in pMHCs to enhance pre-existing contacts and activates new interactions at the TCR-pMHC binding interface to resist bond dissociation under force, resulting in TCR-pMHC catch bonds and T cell activation. Intriguingly, cancer-associated somatic mutations in HLA-A2 that may restrict these conformational changes suppressed TCR-pMHC catch bonds. Structural analysis also indicated that HLA polymorphism might alter the equilibrium of these conformational changes. Our findings not only reveal critical roles of force-induced conformational changes in pMHCs for activating TCR-pMHC catch bonds but also have implications for T cell-based immunotherapy. Wu et al. report that a dynamic structural mechanism of mechano-chemical coupling for TCR antigen recognition—that is, mechanical force-induced conformational changes in the agonist peptide-MHC-I—allosterically activates TCR-pMHC-I catch bonds to determine TCR antigen recognition and trigger T cell signaling.

The earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ-CD8 interactions require CD8-MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck.

The TCR integrates forces in its triggering process upon interaction with pMHC. Force elicits TCR catch-slip bonds with strong pMHCs but slip-only bonds with weak pMHCs. We develop two models and apply them to analyze 55 datasets, demonstrating the models’ ability to quantitatively integrate and classify a broad range of bond behaviors and biological activities. Comparing to a generic two-state model, our models can distinguish class I from class II MHCs and correlate their structural parameters with the TCR/pMHC’s potency to trigger T cell activation. The models are tested by mutagenesis using an MHC and a TCR mutated to alter conformation changes. The extensive comparisons between theory and experiment provide model validation and testable hypothesis regarding specific conformational changes that control bond profiles, thereby suggesting structural mechanisms for the inner workings of the TCR mechanosensing machinery and plausible explanations of why and how force may amplify TCR signaling and antigen discrimination.